Hello everyone,
I am overexpressing a gene in Zebrafishes, by using some neural crest cell promoter, through microinjection. I do see some phenotype, but so far, I have seen it in a transgenic line background, and not on any wildtype. Also, the gene is tagged with EGFP, while in the controls, I see beautiful ncc fluorescing in green, in my experimentals, I do not see GFP expression, it fits in with my hypothesis that overexpression of this gene is affecting ncc and GFP expression.I was wondering if anyone could advise me on how to validate my overexpression. I tried making a construct withcrystallin promoter CFP tag,. so that all the injected embryos get blue eyes, but if they lack GFP cells, then, these are overexpressing my gene, and lack of GFP is because of this. Unfortuantely, I could not get a clone for this. qPCR to confirm overeexpression is difficult as ncc's are quite a limited number of cells, and we do not have any experience in their isolation. Antibody staining with antiGFP does not help much, as I hardly see any GFP positive cells in my experimental. Please let me know if you have anything in mind.
Thanks.
Hi,
The goal standard to confirm expression would be a western blot but I assume you don't have the antibody for your gene of interest. However, I actually think that qPCR to confirm overexpression can be a good alternative. It shouldn't be extremely difficult as it is a very sensitive technique (that can potentially work with a cell, see kit "CellDirect One-Step qRT-PCR" from Invitrogen).
Good luck
Dear Francesca,
I understand your point, and I agree with it too. the problem is that isolation of neural crest cells is not easy, we are talking of less than 100 cells in my case, and establishing the protocol requires a lot of time. I am running short of time now. I am looking for a shorter route this time.
Hi Nivedita - have you considered using a vector containing an IRES (an internal ribosome entry site)?
The you could then run two genes off a signal promoter. One could be (our gene) EGFP and one just RFP
i.e. (NCC promoter)--- gene X---IRES---RFP
You could then use the RFP signal in the control vs the mutants for comparison of expression. As the RFP protein is being generated from the same RNA as Gene X
if RFP is being expressed the RNA for gene X is at least present.
There are some caveats for this though
1) If Gene X is killing the NCC cells you won't get RFP expression (as the cells will be dead)
2) You always get weaker expression of the gene after the IRES than you do for the gene up-stream on the RNA strand (so RFP fluorescence might be weak).
Hope this helps/makes sense.
Gary
Dear Gary,
Many thanks for your answer. I should have been a little more careful in my description. My construct is actually promoter_gene_IRES_EGFP, while the control is promoter_IRES_EGFP. I am using IRES in both the constructs. And as you rightly pointed out, the caveat is that my gene might be killing NCCs and hence no GFP expression.
I appreciate your logic though.
Ah - thanks - at least great minds think alike!
I'm afraid then the only suggestion I have is that you fix the embryo's after injection/expression and then section them for IHC/ICC.
You could then use antibodies to NCC cells and to EGFP.
Once you identified the cells this way you could also get a nice tissue/cell morphology.
Sorry I can't be more help!
Gary
Hi Nivedita,
Your usage of IRES-EGFP is a good idea, however, keep in my mind that having a CDS in front of the IRES will affect EGFP expression level, you may want to consider a better control which has a different gene that presumably doesn't affect NCC when over expressed in front of the IRES. To validate that at least your gene of interest got expressed in the NCC lineage, you can try fix embryos at different stages and stained for NCC markers by either ISH or antibody staining. Another thought is that can you tag your gene of interest with a small tag that won't affect its function so that you can directly track the cells expressing the fused protein? Good luck!
Hi! This is maybe a stupid answer but....like Wei Li said, maybe add a flag tag after your gene and then, with a pool of injected embryos do a western blot and use a anti flag antibody! I'm doing the same for my project, but of course if you're expressing your gene in few cells maybe you'll need 40-50 embryos to see something in western blot!
Hi,
I am a frog biologist.
You have the microinjector and cDNA in the expression vector. I think transgenic is good idea when you need see the derivatives from NCC until very late stages, however, you can just inject mRNA of your gene into embryos, instead of promoter constructs just to see the phenotypes.
You can analyze NCC marker by in situ hybridization.
If NCC are killed by the overexpression, NCC marker will be gone, and only fluorescent dextran or whatever lineage marker will remain.
RNA is less toxic than DNA, and if the construction is okay, I'm sure the overexpression is okay. Most importantly, if your gene kills NCC, you don't need to make transgenic.
Oh, P2A cleaved GFP is an alternative idea. The transcript is translated as a single chunk of fusion protein, and later P2A cleavage site divide the proteins in two.
As long as you could see GFP the other half of protein must be there.
Alternative ideas
1. If your gene X is transcription factor, you can make GeneX-GR(hormone inducible)
2. NCC prom--loxP stop x3 loxP--your gene p2A-GFP/Crstallin-CFP x tet ON-Cre
Saying is much easier than doing:-)
Good Luck!!
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